Saita T, Fujiwara K, Kitagawa T, Mori M, Takata K
Faculty of Hospital Pharmacy, Saga Medical School, Japan.
Cancer Chemother Pharmacol. 1990;27(2):115-20. doi: 10.1007/BF00689094.
A highly sensitive enzyme-linked immunosorbent assay (ELISA) for etoposide (EP) was developed, which is capable of accurately measuring as little as 40 pg EP/ml. Anti-EP sera were obtained by immunizing rabbits with EP conjugated with mercaptosuccinyl bovine serum albumin (MS.BSA) using N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM) as a heterobifunctional coupling agent. An enzyme marker was similarly prepared by coupling EP with beta-D-galactosidase (beta-Gal; EC 3.2.23) via DPEM. This ELISA was specific for EP and showed a very slight cross-reactivity with its major metabolite, cis-hydroxy acid of EP (0.91%), but none with 4'-demethylepipodophyllotoxin and drugs commonly used with EP in combination chemotherapy for cancer treatment. The values for EP concentration detected by this assay were comparable with those detected by the high-performance liquid chromatography (HPLC) method. However, the ELISA was about 1,250 times more sensitive in detecting EP at lower concentrations. Using this assay, drug levels were easily determined in the blood and urine of rats for 7 h after i.v. administration of EP at a single dose of 3 mg/kg. Due to its sensitivity and specificity for EP, the ELISA should prove to be a valuable new tool for use in clinical pharmacological studies.
开发了一种用于依托泊苷（EP）的高灵敏度酶联免疫吸附测定（ELISA）方法，该方法能够准确测量低至40 pg EP/ml的含量。通过使用N-[β-(4-重氮苯基)乙基]马来酰亚胺（DPEM）作为异双功能偶联剂，用与巯基琥珀酰牛血清白蛋白（MS.BSA）偶联的EP免疫兔子，获得了抗EP血清。通过DPEM将EP与β-D-半乳糖苷酶（β-Gal；EC 3.2.23）偶联，同样制备了酶标记物。这种ELISA对EP具有特异性，与其主要代谢物EP的顺式羟基酸有非常轻微的交叉反应（0.91%），但与4'-去甲基表鬼臼毒素以及在癌症联合化疗中与EP常用的药物无交叉反应。该测定法检测到的EP浓度值与高效液相色谱（HPLC）法检测到的值相当。然而，ELISA在检测较低浓度的EP时灵敏度约高1250倍。使用该测定法，在以3 mg/kg的单剂量静脉注射EP后7小时内，很容易测定大鼠血液和尿液中的药物水平。由于其对EP的灵敏度和特异性，ELISA应被证明是临床药理学研究中一种有价值的新工具。
